The intent of this project is to develop an effective dendritic cell (DC) vaccine for solid tumors. While DC vaccines can induce potent peripheral T-cell immunity, their antitumor effects have been limited. This is most likely due to the presence of immunosuppressive cells within the tumor, such as regulatory T cells (Tregs), as well as tumor sporting stroma. While vaccine strategies targeting Tregs or cancer associated fibroblasts (CAFs, the major component of tumor stroma) have produced complete responses in animal models, tumors inevitably recur, as effector T-cell functions are eroded and/or subverted. We now hypothesize that by targeting not only the tumor and Tregs, but also CAFs, T-cell effector function will be retained for longer and the local 'tumor promoting heaven' will be destroyed resulting in sustained tumor remissions. We further hypothesize that the potent immunostimulatory environment created in the tumor will result in epitope spreading as newly activated antigen presenting cells within the tumor stroma 'cross-present' dying tumor cells. To test these hypotheses we will take advantage of our preliminary studies in which we have demonstrated that silencing the negative regulator A20 in DC can overcome Treg mediated immune suppression resulting in potent antitumor responses in the B16 model. In addition, we have shown that an A20-silenced DC vaccine can induce potent immune responses against fibroblast activating protein (FAP) expressed on the cell surface of CAFs. Using the B16 model, we will evaluate in Aim 1 if a DC vaccine coexpressing A20-shRNA, FAP and TRP2 (DC-shA20-FAP-TRP2) reverses the suppressive tumor microenvironment and facilitates T-cell infiltration into tumors, and determine in Aim 2 the potency of our vaccine to induce bystander T-cell responses (epitope spreading), leading to elimination of tumor antigen loss variants. In Aim 3 we will then evaluate the efficacy and safety of our vaccine in a spontaneous murine Hgf-Cdk4R24c melanoma model. The proposed study is highly innovative since it will test for the first time the ability of genetically modified DCs to induce T-cell responses against cancer cells and their supporting stroma, while concomitantly overcoming Treg-mediated immunosuppression in a single vaccine formulation. If this pre-clinical approach is successful and clinical validation appears justified, we have the resources to prepare vector and cellular components that conform to the appropriate Good Manufacturing Practices safety standards, and will seek separate funding for such a study.